The Role of Eosinophil-Derived Neurotoxin and Vascular Endothelial Growth Factor in the Pathogenesis of Eosinophilic Asthma

Asthma is a chronic complex pulmonary disease characterized by airway inflammation, remodeling, and hyperresponsiveness. Vascular endothelial growth factor (VEGF) and eosinophil-derived neurotoxin (EDN) are two significant mediators involved in the pathophysiology of asthma. In asthma, VEGF and EDN levels are elevated and correlate with disease severity and airway hyperresponsiveness. Diversity in VEGF polymorphisms results in the variability of responses to glucocorticosteroids and leukotriene antagonist treatment. Targeting VEGF and eosinophils is a promising therapeutic approach for asthma. We identified lichochalcone A, bevacizumab, azithromycin (AZT), vitamin D, diosmetin, epigallocatechin gallate, IGFBP-3, Neovastat (AE-941), endostatin, PEDF, and melatonin as putative add-on drugs in asthma with anti-VEGF properties. Further studies and clinical trials are needed to evaluate the efficacy of those drugs. AZT reduces the exacerbation rate and may be considered in adults with persistent symptomatic asthma. However, the long-term effects of AZT on community microbial resistance require further investigation. Vitamin D supplementation may enhance corticosteroid responsiveness. Herein, anti-eosinophil drugs are reviewed. Among them are, e.g., anti-IL-5 (mepolizumab, reslizumab, and benralizumab), anti-IL-13 (lebrikizumab and tralokinumab), anti-IL-4 and anti-IL-13 (dupilumab), and anti-IgE (omalizumab) drugs. EDN over peripheral blood eosinophil count is recommended to monitor the asthma control status and to assess the efficacy of anti-IL-5 therapy in asthma.


Eosinophilic Asthma
Asthma is one of the most common chronic pulmonary diseases and affects approximately 400 million people worldwide with a gradually increasing incidence [1]. In 2019, the global prevalence of asthma was 9.8%. It is estimated that over 260 million people have poorly controlled asthma [2,3]. Asthma is an inflammatory disease characterized by respiratory symptoms such as wheezing, chronic cough, chest tightness, and shortness of breath that results from bronchial hyperreactivity and inflammation [4,5]. Allergens, obesity, tobacco smoke, exercise, cold air, genetic mutations, and systemic eosinophilia are factors that induce chronic inflammation leading to airway hyperresponsiveness [6,7]. Chronic inflammation causes airway swelling, remodeling, and excessive mucus secretion.
Th2 cells and the cytokines they secrete (IL-4, IL-5, IL-13, and IL-9) are responsible for most of the pathological changes seen in asthma [23]. Once the sensitization phase has passed, the repeated arrival of the allergen in the lower airways results in the induction of mast cells by IgE to release mediators such as leukotrienes (LTs), histamine, and interleukins. These mediators irritate airway smooth muscle and induce bronchospasm [23][24][25]. In addition, IL-5 causes the production and chemotaxis of eosinophils in the lungs [26]. IL-13 sensitizes airway smooth muscle to spasm, stimulates epithelial cells to secrete mucus, and induces fibrosis [9,27].
Based on the state of Th2 inflammation, the disease can be divided into two groups: Th2-high and Th2-low asthma. Th2-high asthma is characterized by eosinophilic airway inflammation (eosinophilic asthma), which is associated with an increased number of eosinophils in the blood, while Th2-low asthma includes neutrophilic asthma and paucigranulocytic asthma. Although asthma is divided into Th2-high and Th2-low, both asthma phenotypes can occur simultaneously in some patients [21,22,28].

Th1 Response
Th1 cells mainly secrete IL-2 and interferon-γ (IFN-γ) to protect the body from intracellular bacteria and viruses [29]. Epithelial damage promotes the maturation of Th1 cells and the secretion of Th1 cytokines, including tumor necrosis factor (TNF-α) and IFN-γ.
TNF-α interacts with IL-17 cytokines to promote neutrophil recruitment. In addition, TNF-α enhances airway smooth muscle contraction [30,31]. It has been proven that IFN-γ secretion by Th1 cells is associated with the suppression of secretory leukocyte protease inhibitor, which is associated with airway hyperresponsiveness and pathological changes in the lungs [32,33].

Th17 Response
Th17 cells produce both IL-17 and IL-22, which contribute to the development of asthma [6]. IL-17, through the activation of epithelial cells, fibroblasts, and smooth muscle cells, contributes to airway remodeling [34]. Additionally, IL-17A increases bronchial smooth muscle contraction [6,35]. However, it has been suggested that IL-17 is important for maintaining epithelial integrity; thus, IL-17 may play a protective role against asthma [36].
The asthma phenotype should be taken into account when choosing the appropriate treatment as the prognosis and response to drugs in eosinophilic and neutrophilic asthma are different.

Aim of the Study
Growing evidence suggests that EDN is more applicable than eosinophil blood count and ECP in evaluating disease severity [37]. VEGF may be an underappreciated proinflammatory factor contributing to asthma. Thus, the aim of this paper was to evaluate the biological function and role of EDN and VEGF in asthma pathogenesis. We also review the current knowledge about putative approaches of anti-VEGF and anti-eosinophil drugs in asthma therapy.

VEGF VEGF in Asthma Pathogenesis
Vascular endothelial growth factor (VEGF) belongs to a family of proteinaceous growth factors and is one of the most potent inducers of vasculogenesis and angiogenesis [38][39][40]. The cells involved in the synthesis and action of vascular endothelial growth factor are tumor cells, bronchial and lung epithelial cells, smooth muscle cells, vascular endothelial cells, macrophages, neutrophils, and Th2 lymphocytes. VEGF's main targets are vascular endothelial cells, but its activity also includes monocytes, macrophages, embryonic stem cells, and neurons [41,42]. VEGF subfamily ligands' most prominent structural feature is a cystine junction composed of three intertwined disulfide bridges [40,43].
The VEGF subfamily consists of VEGF-A to VEGF-F and placental growth factor (PlGF) ( Figure 1). However, we focus on VEGF-A due to its crucial role in angiogenesis, vasodilatation, the release of nitric oxide, and enhancing the chemotaxis of macrophages and granulocytes [39,44]. Hypoxia and inflammation stimulate hypoxia-inducible factors (HIFs), which can increase the activity of various proangiogenic factors through the HIF-1α pathway, as well as VEGF-A, becoming one of its primary regulators [45][46][47]. In addition to hypoxia-inducible factors (HIFs), there are other regulators of VEGF expression. One group with this function comprises growth factors, including epidermal growth factor, transforming growth factors (TGF) α and β, insulin-like growth factor-1, fibroblast growth factor, and platelet-derived growth factor (PDGF). During inflammation, interleukins (e.g., interleukin (IL)-1β or IL-6) are secreted, increasing VEGF expression in many cell types. VEGF modulation can also occur during oncogene mutations due to oncogenes (Ras) or tumor necrosis factor (TNF-α) [48]. The gene for VEGF-A is located on chromosome 6 and undergoes alternative splicing of exons to produce isoforms such as VEGF121, VEGF165, VEGF189, and VEGF206, of which VEGF165 is the most common isoform in tissues [39]. VEGF165, VEGF189, and VEGF206, of which VEGF165 is the most common isoform in tissues [39]. VEGF-B is involved in neovascularization occurring during embryonic development and in the progression of cancerous tumors. VEGF-C increases vascular permeability and the formation of lymphatic vessel networks [44]. VEGF-D is a tumor angiogenesis factor and promotes EC proliferation. It also modulates the abundance of lymphatic vessels in specific tissues during embryonic development. Understanding and confirming its other functions requires further research [44,49]. Placental growth factor (PLGF) may be involved in the growth and maintenance of pregnancy and wound healing [44]. Increased VEGF expression correlates with poorer clinical outcomes in many diseases; however, VEGF also has many possible therapeutic uses [39,50].
The VEGF receptors (VEGFRs) are members of the type III transmembrane tyrosine kinases (TKs) superfamily of receptors. For example, they are structurally related to PDGFR, IR, IGFR, or FGFR receptors [51,52]. VEGFRs consist of three subtypes: VEGFR-1 and VEGFR-2, both mainly found on vascular endothelial cells, and VEGFR-3, primarily located on lymphatic endothelial cells [52,53]. Structurally, VEGFRs are similar to each other. They have an extracellular domain consisting of seven Ig homology domains (D1-D7), a transmembrane domain, a juxtamembrane regulatory domain, and a tyrosine kinase domain [54,55].
The mechanism of VEGFR activation involves ligand-induced dimerization of the extracellular domain, followed by tyrosine autophosphorylation in the intracellular kinase domain to generate downstream signaling. The D2 domain serves mainly for ligand binding by VEGFR, with VEGFR-3 using the D1 domain for this purpose. The D3 serves to increase binding affinity. The D4-7 domains are involved in generating structural changes that are essential for receptor dimerization and activation [56,57]. Experiments on the receptor's kinase domain have shown that domains 4-7 are not essential for signaling [58]. While VEGF-A binds with high affinity to VEGFR-1, it signals blood vessel development mainly through VEGFR-2 [54]. Conversely, VEGF-B and PIGF bind only to VEGFR-1 [59]. The growth factors primarily responsible for the development of lymphatic vessels are VEGF-C and VEGF-D, which signal especially through VEGFR-3 [60]. Studies conducted by Tammel et al. have shown that despite expression mainly on lymphatic vessels, VEGF-B is involved in neovascularization occurring during embryonic development and in the progression of cancerous tumors. VEGF-C increases vascular permeability and the formation of lymphatic vessel networks [44]. VEGF-D is a tumor angiogenesis factor and promotes EC proliferation. It also modulates the abundance of lymphatic vessels in specific tissues during embryonic development. Understanding and confirming its other functions requires further research [44,49]. Placental growth factor (PLGF) may be involved in the growth and maintenance of pregnancy and wound healing [44]. Increased VEGF expression correlates with poorer clinical outcomes in many diseases; however, VEGF also has many possible therapeutic uses [39,50].
The VEGF receptors (VEGFRs) are members of the type III transmembrane tyrosine kinases (TKs) superfamily of receptors. For example, they are structurally related to PDGFR, IR, IGFR, or FGFR receptors [51,52]. VEGFRs consist of three subtypes: VEGFR-1 and VEGFR-2, both mainly found on vascular endothelial cells, and VEGFR-3, primarily located on lymphatic endothelial cells [52,53]. Structurally, VEGFRs are similar to each other. They have an extracellular domain consisting of seven Ig homology domains (D1-D7), a transmembrane domain, a juxtamembrane regulatory domain, and a tyrosine kinase domain [54,55].
The mechanism of VEGFR activation involves ligand-induced dimerization of the extracellular domain, followed by tyrosine autophosphorylation in the intracellular kinase domain to generate downstream signaling. The D2 domain serves mainly for ligand binding by VEGFR, with VEGFR-3 using the D1 domain for this purpose. The D3 serves to increase binding affinity. The D4-7 domains are involved in generating structural changes that are essential for receptor dimerization and activation [56,57]. Experiments on the receptor's kinase domain have shown that domains 4-7 are not essential for signaling [58]. While VEGF-A binds with high affinity to VEGFR-1, it signals blood vessel development mainly through VEGFR-2 [54]. Conversely, VEGF-B and PIGF bind only to VEGFR-1 [59]. The growth factors primarily responsible for the development of lymphatic vessels are VEGF-C and VEGF-D, which signal especially through VEGFR-3 [60]. Studies conducted by Tammel et al. have shown that despite expression mainly on lymphatic vessels, VEGFR-3 is also involved in regulating blood vessel network formation, and its blockade may contribute to the efficacy of anti-angiogenic therapies [61].
An important function of VEGFR-1 is its role as a decoy for VEGF-A, serving as an endogenous anti-angiogenic factor by reducing its bioavailability to target cells. VEGFR-1, also produced in the placenta during pregnancy, binds to placental growth factor (PIGF) in the form of both VEGF/PIGF heterodimers and PIGF homodimers, which is important in, among other things, pre-eclampsia during pregnancy [62][63][64]. Numerous studies have also shown that under specific conditions, after stimulation by PIGF, VEGFR-1 can heterodimerize with VEGFR-2, transactivating it and positively regulating angiogenesis. In addition, PIGF can increase the expression of VEGF, further enhancing its positive effects on angiogenesis [65][66][67].
Important co-receptors for VEGF are neuropilins (NRPs). They are divided into NRP-1, expressed in arterial endothelial cells, and NRP-2, expressed in the endothelial cells of veins and lymphatic vessels. They are active protein receptors and can regulate neurogenesis and angiogenesis. The best-known binding is that of VEGF-A (specifically, VEGF-A165 or VEGF-A189) with NRP-1 in complex with VEGFR-1 and VEGFR-2. This results in forming a VEGF-A/VEGFR/NRP1 complex. Such a combination induces signal transduction downstream of the receptor, stimulating it and contributing to the activation of angiogenesis [48,[68][69][70].
Research on the role of VEGF in the pathogenesis of bronchial asthma has shown that it can stimulate allergic inflammation in the bronchial tree and contribute to the remodeling processes. Numerous studies have also proven that asthmatic patients, including children, have significantly elevated levels of VEGF [71][72][73][74]. The study by Hoshino et al. demonstrated an increased number of vessels per unit area with no significant difference in mean vessel size in asthmatic patients compared to their number and size in non-asthmatic control subjects. Furthermore, they proved a correlation between the area of vessels and the amount of VEGF in the airways in asthmatic subjects [75]. The results of a study by Zhang et al. show that the highest levels of VEGF were observed in individuals during the acute phase of asthma, with lower levels in patients with stable asthma, and the lowest levels in the control group. In addition, by determining the sputum VEGF concentration in subjects with mild, moderate, or severe asthma, they proved a significant correlation between the VEGF-level increase and aggravation of the patient's condition [76]. A different study confirmed the above results by showing elevated expression levels of both VEGF and its receptor VEGFR1 in the bronchial tissue of asthmatic patients. These correlated well with the extent of airway remodeling and airway hyperresponsiveness and were negatively correlated with lung function data. Patients showed submucosal gland hyperplasia, increased smooth muscle mass, increased reticular basement membrane thickness, subepithelial fibrosis, and neovascularization, compared with control subjects [77].
Mucins are responsible for mucus viscoelasticity, and their increased presence in the airways is due to mucus hypersecretion in asthma. MUC5AC is the main mucin glycoprotein overproduced in asthma. Studies have shown that VEGF increases MUC5AC mRNA expression in a dose-and time-dependent manner through the activation of RhoA kinases [78]. Asthma is connected with increased airway smooth muscle mass [79]. VEGF significantly affects matrix metalloproteinases MMP-1 and MMP-9 with a lesser impact on MMP-3. The endothelial growth factor increases their production, and studies suggest that this contributes to smooth muscle migration in angiogenesis [80].VEGF has also been confirmed to increase the expression of disintegrin and metalloproteinase (ADAM)-33, a factor that plays a significant role in the pathophysiology of asthma, as well as the proliferation of airway smooth muscle cells through activation of the VEGFR2/ERK1/2 pathway [81].
Genetic analyses of the airway smooth muscle cells in bronchial asthma patients have revealed epigenetic modifications of histones. The histone methylation pattern described during the study suggests that the VEGF promoter in asthmatic patients is more active and is also deprived of the repression mechanism present in the promoter of healthy patients. This mechanism involves the induction of histone H3K9 methylation by G9A recruitment, which results in reduced RNA pol II binding and reduced binding of Sp1, the VEGF promoter. This results in overexpression of the endothelial growth factor [82]. VEGF levels are also affected by claudin-1, whose elevated levels were observed in patients with asthma and a mouse model of asthma-like airway inflammation. Overexpression of claudin-1 Cells 2023, 12, 1326 6 of 26 resulted in increased proliferation of airway smooth muscle cells and significantly elevated levels of VEGF [83]. A study by de Paulis et al. demonstrated the possible influence of basophils on angiogenesis and inflammation through the synthesis and release of various VEGF isoforms from the cytoplasmic secretory granules of basophils. They also showed that some VEGF-A isoforms could function as chemotactors for basophils. Hence, this indicates that there is an essential autocrine loop that may participate in the development of asthma [84]. The effect of basophils on VEGF due to the previously mentioned hypoxiainducible factor has also been demonstrated. The study showed that basophil activation by IgE results in the accumulation of the α subunit of hypoxia-inducible factor 1α (HIF-1α), which correlates with an increase in HIF-1α mRNA, as well as VEGF. Inhibition of HIF-1α expression in basophils showed that this protein is essential for the expression of vascular endothelial growth factor (VEGF) mRNA and, consequently, its release [85].
As mentioned above, interleukins also affect the concentration of VEGF in the body. For instance, interleukin 32 (IL-32) has been shown to affect VEGF levels. The study showed that silencing the previously mentioned interleukin in normal human bronchial epithelial cells increased VEGF secretion. Moreover, high levels of IL-32 correlated with better responses to treatment in patients with bronchial asthma. Accordingly, this implies that an adequate amount of IL-32 reduces endothelial growth factor production and angiogenesis [73]. Other studies have also proven that elevated IL-25 levels in asthma contribute to angiogenesis, at least in part by increasing VEGF/VEGF receptor expression in endothelial cells through the PI3K/Akt and Erk/MAPK pathways [86]. Interestingly, interleukin-5 has been shown to have anti-angiogenic potential. It has been shown to significantly inhibit vascular formation, including VEGF-induced endothelial cell proliferation, migration, and tube formation, acting through STAT5. Its blockade abolished the anti-angiogenic effect of IL-5 [87]. However, interleukin 33, whose elevated levels are induced by hypoxia, may partially increase the expression of HIF-1α and VEGF to initiate blood vessel remodeling [88]. Studies have confirmed the link between VEGF and the inflammatory response stimulated by Th2 lymphocytes. Furthermore, VEGF is a potent stimulator of inflammation and dysregulation while increasing the antigen sensitivity and inflammation of Th2 lymphocytes and increasing the number and activation of local dendritic cells. Studies have also revealed that VEGF production plays a significant role in forming Th2 cytokines and that epithelial cells and Th2 cells are potent producers of VEGF in the antigen-affected lung [89].
One paper summarized the relationships between micro-RNA, VEGF, and asthma. The meta-analysis results showed that some miRNAs have pro-angiogenic properties and can be stimulated by pro-angiogenic stimuli such as VEGF, among others. As an example, the expression of pro-angiogenic miR-130a or miR-132 increased under the influence of VEGF. Contrarily, miR-126 was described as having an anti-angiogenic effect, and its downregulation increased VEGF activity. The role and possible benefits of miRNAs in asthma are not yet well understood and require further and more thorough research [90].
It was also discovered that rhinovirus infection causes VEGF production in human airways, mainly by fibroblasts, which is further exacerbated in an atopic environment. The angiogenesis stimulated by rhinoviruses may cause the condition of patients with asthma to become worse [91,92].
Research conducted in China has revealed a correlation between VEGF polymorphisms and the prevalence of asthma in the Chinese Han population. The rs3025020 polymorphism of the VEGF gene was found to be related to asthma, and the frequency of the T allele in the asthma group was significantly higher than in the control group. Findings suggest that the VEGF rs3025020 polymorphism plays a significant role in cell proliferation and inappropriate VEGF-induced angiogenesis related to asthma [93]. Studies evaluated the association between VEGF polymorphisms and childhood asthma, lung function, and airway reactivity in two populations. In both of them, the rs833058 polymorphism was associated with asthma. It was also linked with increased airway reactivity among the subjects. Furthermore, it impacted the decrease in the ratio of forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC) over~4.5 years of follow-up of the subjects [94]. In addition, it was shown that the presence of the rs3025028 polymorphism of the VEGF-A gene associated with airway function parameters measured in childhood correlates with the persistence of the effect after adulthood [95]. Moreover, subsequent studies of VEGF-A have shown a correlation between its gene polymorphisms and response to therapy with inhaled corticosteroids or leukotriene receptor antagonists (LTRAs). The AA genotype of the rs2146323 polymorphism correlated with a better response to corticosteroid therapy and a worse response to LTRA therapy. Furthermore, the rs833058 polymorphism was associated with a better response to episodic LTRA therapy [96].

EDN in Asthma Pathogenesis
EDN (RNase2) belongs to the ribonuclease A superfamily [97]. EDN is a single-chain polypeptide with a molecular weight of 18.6 kDa. A comparison of the partial N-terminal amino acid sequences of EDN and ECP showed 67% identity and structural homology to pancreatic ribonuclease (RNase) [98,99]. EDN expression has been detected in the highest concentration in eosinophils. EDN expression was also found in monocytes, dendritic cells, basophils, and neutrophils [100,101]. In addition to its neurotoxic effects, EDN has been shown to have antiviral activity, particularly against the respiratory syncytial virus. As a result, it participates in the defense of the upper bronchial region against viral infection. Hence, it is suggested that EDN is involved in host defense against single-stranded RNA viruses [102][103][104][105]. In addition, EDN is associated with allergic airway inflammation [106]. In summary, EDN is a multifunctional mediator with cytotoxic, antiviral, and chemotactic effects on DCs [102] (Figure 2).

EDN vs. Blood Eosinophil Counts in Asthma Control Status
Eosinophils play a central role in allergic diseases, so direct measurement of eosinophilic inflammation is essential for diagnosing, treating, and monitoring patients with asthma. Over recent years, specific markers have been identified and used to identify eosinophil activity and turnover. One of the markers has been EDN, which has been studied in a number of inflammatory diseases [114,115]. This neurotoxin, as a product of eosinophil degranulation, is attracting much attention as a new biomarker for diagnosing and monitoring asthma in children and adults [37]. In patients with asthma, levels of type-2 instructional cytokines (IL-33 and IL-25) and effector cytokines (IL-4, IL-5, and IL-13) are elevated in the airway mucosa, associated with impaired antiviral immunity. In a study conducted in 2022, it was shown that expo- sure to eosinophils or eosinophil supernatants inhibited RV-induced IFN-α secretion by dendritic cells [107]. Eosinophil-secreted factors such as TGF-β and EDN suppressed the antiviral response by inhibiting CXCL10 and IFN-α production by dendritic cells (pDCs). The rhinovirus-stimulated pDCs secreted significantly less IFN-α and CXCL10 when cocultured with eosinophils. Accordingly, this provided evidence that eosinophils attenuate the antiviral immunity of pDCs [108].
EDN is released from eosinophil granules after activation by cytokines (including IL-5, IL-11, and IL-33) and other proinflammatory mediators [109]. RNAase2 is selectively chemotactic for dendritic cells (DCs). Additionally, it induces the activation of mitogenactivated protein kinase p42/44 (MAPK) in DCs [102,110]. EDN can also induce the production of proinflammatory cytokines in monocytes/macrophages and the maturation of dendritic cells through Toll-like receptor 2 (TLR2) [111]. Studies suggest that eosinophilderived neurotoxin also plays a role in allergic diseases [104]. Therefore, EDN promotes the allergic response by activating dendritic cells. EDN levels are closely related to Th2 inflammation [19]. Eosinophil-derived neurotoxins have been classified as alarmins, which are endogenous mediators rapidly released by cells of the host's innate immune system in response to infection. Alarmins can activate antigen-presenting cells and enhance the immune system response [111][112][113].

EDN vs. Blood Eosinophil Counts in Asthma Control Status
Eosinophils play a central role in allergic diseases, so direct measurement of eosinophilic inflammation is essential for diagnosing, treating, and monitoring patients with asthma. Over recent years, specific markers have been identified and used to identify eosinophil activity and turnover. One of the markers has been EDN, which has been studied in a number of inflammatory diseases [114,115]. This neurotoxin, as a product of eosinophil degranulation, is attracting much attention as a new biomarker for diagnosing and monitoring asthma in children and adults [37].
In connection with the fact that EDN is secreted almost exclusively by eosinophils, this indicator may directly reflect exacerbations in eosinophilic inflammation [116].
Studies have suggested that airway inflammation associated with asthma exacerbation is characterized by an increase in eosinophils and an increase in eosinophil degranulation in the airways [37,117].
Measurement of eosinophils in sputum has been a widely used test to assess the status of patients with asthma. However, eosinophil levels may not always accurately represent the cellular state of the asthmatic airways [118]. It has been proven that the measurement of eosinophils in sputum or blood does not sufficiently correlate with the severity of airway inflammation in asthma, e.g., eosinophilia develops several weeks before an exacerbation of the disease [119,120]. It has been suggested that eosinophil secretory activity is equal to the product of the eosinophil concentration, and mediators, e.g., EDN and ECP, could be critical markers of disease severity [37,115,121].
Regardless of the asthma phenotype, when eosinophils are activated, they release EDN from their granules [37,100]. Recent studies have confirmed the efficacy of EDN as a marker of eosinophilic inflammation to monitor and treat asthma [116,[122][123][124]. It was demonstrated that EDN could act as a biomarker reflecting lung function and as a biomarker positively correlated with asthma severity [114,125]. In a study by Kim et al., EDN levels were higher and lung function was decreased in patients with eosinophilic asthma exacerbations [125].
It has been shown that the EDN concentration test has high accuracy, and applicability on small-volume samples of specimens such as sputum, serum, and urine [126]. EDN measurement is an affordable test with practical application in the daily diagnosis, treatment, and monitoring of several eosinophil-related disorders, such as asthma and recurrent wheezing bronchiolitis caused by a respiratory syncytial virus (RSV) [114,127]. Serum levels of EDN were shown to be significantly different between patients with controlled and uncontrolled asthma [128]. A study by An et al. showed that the mean serum levels of EDN in the group with uncontrolled asthma were higher than those in the group with controlled asthma and healthy patients. Serum EDN levels were correlated with the total eosinophil count (TEC), but a receiver operating characteristic (ROC) curve analysis showed that serum EDN levels were significantly better at predicting uncontrolled asthma. This study demonstrates that EDN predicts asthma control better than blood eosinophil count [121]. In contrast, however, a study by Gon et al. did not indicate a significant correlation between blood eosinophil count or serum EDN with lung function and symptom scores in patients with asthma [129].
The diagnosis of allergic disease in young children is a difficult task. Examining airway function in children is particularly difficult because they cannot participate in various functional tests. It should be noted that poor asthma control leads to poor quality of life for children and their caregivers [130,131]. In the pediatric population, EDN may be a promising biomarker in distinguishing patients with persistent wheezing from those with wheezing caused by respiratory infections, and may be an aid in diagnosing school-age asthma [129,132].
It has been reported that children in the acute phase of asthma may have higher serum EDN levels than those in the stable phase, and in contrast to the TEC, serum EDN levels may be a predictor of asthma severity [124,133]. In addition, in children, EDN levels were significantly higher in the atopic asthma group than in the non-atopic asthma group or control patients [134].

Anti-VEGF
Given the above data, one can speculate that anti-VEGF treatment would benefit asthma therapy (Table 1). Glucocorticoids are the most effective medications currently available for the treatment of asthma. They act primarily as an anti-inflammatory and partially reduce the airway hyperresponsiveness characteristic of the condition [135,136]. For example, the use of budesonide in therapy significantly suppressed the spontaneous release of VEGF, and the production of VEGF increased by IL-4, IL-5, IL-13, TGF-β, or IL-1β [137]. Other studies have also confirmed that treatment with budesonide and formoterol reduces the expression of both VEGF and VEGFR1, which correlates with reduced airway remodeling in patients with asthma [77]. The previously mentioned rhinovirus is one of the stimulators of VEGF release. Budesonide has been shown to suppress the chemokines it induces, including VEGF [138].
Montelukast is a selective CysLT1 receptor antagonist used to treat asthma. One study investigated the effect of montelukast on the parameters of irritant-induced asthma induced by the inhalation of chlorine in mice. Montelukast inhibited this increase and effectively blocked the elevation of VEGF and IL-6 concentrations involved in inflammation via the CysLT1 receptor. However, the neutralization of IL-6, but not VEGF-A, attenuated chloride-induced neutrophilia and bronchial tree hyperresponsiveness in the lungs [139].
The study by Türkeli et al. may point to other treatment pathways for asthma. An experimental mouse model of asthma showed that anti-VEGF therapy effectively reduced growth factors and appeared to increase levels of the epithelial barrier proteins E-cadherin and β-catenin. For this reason, the use of anti-VEGF therapy in asthma seems to be a more effective treatment for epithelial barrier reconstruction and remodeling than, e.g., corticosteroid treatment and TNF-α inhibition, which were not effective in increasing E-cadherin and β-catenin levels [140].
Studies were also performed on the effect of licochalcone A on VEGF-induced respiratory smooth muscle cell proliferation. They showed that it inhibits the process, probably by blocking VEGFR2 and ERK1/2 activation and downregulating caveolin-1 [79].
Diosmetin has anti-inflammatory properties and may be another potential drug to treat asthma. Its administration resulted in a significant reduction not only in VEGF levels but also matrix metallopeptidase-9 and transforming growth factor-β1. Accordingly, this suggests a link between reduced airway remodeling and a decrease in these proteins [141].
Bevacizumab is an anti-VEGF monoclonal antibody that also shows potential in asthma treatment applications. Using a mouse model of house dust-mite (HDM)-induced asthma, it was found to reduce airway hyperresponsiveness and inflammation induced by HDM. In addition, it appeared to reduce the release of Th2 cytokines. The effect of bevacizumab administration may be due to the neutralization of VEGF-A and inhibition of VEGFR-2 activation [142]. The effective reduction of inflammation and reduced VEGF release is also supported by more recent studies, which suggest that inhibiting angiogenesis in rats with induced asthma not only suppresses the inflammatory process by blocking VEGF expression but also inhibits the development of new blood vessels and the progression of asthmatic attacks [143].
Another possible treatment option may be silver nanoparticles, which have been shown to reduce VEGF signaling through the PI3K/HIF-1α/VEG pathway, EGFR levels, and MUC5AC expression, while having minimal toxicity [144]. Studies have also shown that silver nanoparticles can have an anti-angiogenic effect. In this case, the effect was confirmed by inducing the death of primary bovine retinal endothelial cells, even in the presence of VEGF, by up to 50%. In the same model, it was also proven that silver nanoparticles inhibit VEGF-induced cell proliferation and migration. In the VEGF-treated material, increased endothelial cell migration and complete wound closure were observed no later than 24 h after treatment, while in the silver-nanoparticle-treated samples, a vast area of the wound remained exposed [145].
The effect of pigment epithelium-derived factor (PEDF) on airway remodeling in chronic allergic asthma was also studied. It was found that PEDF has an inhibitory effect on eosinophil-induced airway inflammation, airway hyperreactivity, and airway remodeling. In addition, mice experienced a significant inhibitory effect on ovalbumin-stimulated VEGF production. PEDF also inhibited VEGF release from IL-1β-stimulated BEAS-2B cells [146].
The next potential drug is epigallocatechin gallate (EGCG). EGCG is the active catechin found in green tea. In a paper by Yang et al., the protective effect of EGCG against HDMinduced asthma was studied in mice. The results showed that it alleviates tissue damage, inflammation, mucus production, and collagen deposition, and reduces M2 macrophage infiltration. The tested compound alleviates asthma symptoms in mice by suppressing HIF-1α/VEGFA-mediated M2 macrophage skewing. Hence, this confirms that restoration of both HIF-1α and VEGFA significantly blocked the protective functions of EGCG [148]. EGCG has also been shown to reduce lung injury and airway remodeling in asthmatic rats exposed to PM2.5 and to have a protective effect on the lungs. This mechanism may be associated with regulation of the HMGB1/RAGE signaling pathway [149].
There have also been studies on mice that have shown a beneficial effect of prolonged azithromycin (AZT) administration on airway remodeling. This is achieved through the PI3K/Akt/mTOR/HIF-1α/VEGF pathway. Reduced airway reactivity and fewer lesions were observed, as confirmed by significantly reduced HIF-1α and VEGF levels [150]. Other studies have also confirmed the beneficial effects of AZT, revealing its anti-angiogenic effect through the p38(MAPK) pathway caused by fibroblast growth factor stimulation [151]. Long-term low-dose azithromycin therapy has been proven to be beneficial in patients with different endotypes of severe asthma. A substantial reduction in the number of asthma exacerbations during therapy and an improvement in their condition were observed. AZT is less toxic than maintenance inhaled corticosteroids, so it could be used before them in severe non-eosinophilic asthma [152]. The effect of AZT on poorly controlled asthma in children has also been studied. The use of AZT in children resulted in improved asthma control and fewer exacerbations without significant side effects. The beneficial effect of AZT was similar in children with both eosinophilic and non-eosinophilic asthma [153].
Other studies have shown that vitamin D inhibits VEGF-induced respiratory smooth muscle cell proliferation by suppressing VEGFR2 and ERK1/2 activation and downregulation of ADAM33, which may be crucial in developing therapies for diseases such as asthma [154].
Studies of the shark-cartilage-derived anti-angiogenic drug Neovastat (AE-941) in the treatment of asthma have also been conducted. Reports have shown its ability to suppress the activity of HIF-2α, one of the hypoxia-induced factors that increase VEGF levels. However, the efficacy and feasibility of Neovastat need to be confirmed by more research [155].
The effect of endostatin/Fc on ovalbumin-induced cellular immunization was examined in a mouse model of asthma. It was shown to inhibit airway hyperreactivity, allergic pulmonary inflammation, ovalbumin-specific IgE production, and lung inflammation mediators. However, data from the study show only partial inhibition of asthma features by the VEGF receptor blockade, indicating that it is not only the result of VEGF antagonism signaling but may also act through other mechanisms [156].
Recently, the effect of melatonin on airway remodeling in asthma has been studied. The results indicate that melatonin significantly reduces airway hyperreactivity, inflammation, and remodeling in a house dust-mite model. Melatonin was also found to significantly inhibit airway smooth muscle cell proliferation, VEGF synthesis, and PDGF-induced cell migration, which may depend on STAT3 signaling. These studies point to the future possibility of using melatonin to treat asthma [157].  [157,174,175] ND-no data; TRPV1-transient receptor potential vanilloid 1; ERK-extracellular signal-regulated kinase; FEV(1)-forced expiratory volume in one second; MAPK-mitogen-activated protein kinase; MUC5AC-mucin 5AC.
EDN levels have been shown to decrease after treatment with budesonide and benralizumab [179]. Benralizumab is a monoclonal antibody directed against interleukin-5Rα, which selectively reduces the number of eosinophils through increased antibody-dependent cellular cytotoxicity. The drug reduces the frequency of severe asthma exacerbations and lowers the daily dose of corticosteroids [180]. In a study, benralizumab used in patients with asthma was shown to reduce blood eosinophil counts and serum EDN and ECP from baseline values [181].
Mepolizumab is also a monoclonal antibody directed against IL-5. It has been shown to reduce the frequency of AE by about half in participants with severe eosinophilia and improve patients' quality of life [182]. A study by Gon et al. showed a significant correlation between the reduction in serum EDN from baseline and improved lung function after omalizumab treatment [129].
Another study of 15 patients with severe eosinophilic asthma showed that after 6month treatment with reslizumab, total eosinophil count (TEC) and EDN decreased with a significant increase in FEV (1). Despite the small study group, these findings suggest that EDN is a valuable biomarker for monitoring anti-IL-5 treatment [178].  [13,177,190,191,196,197 AER-annualized exacerbation rate; FEV(1)-forced expiratory volume in one second; ACQ-Asthma Control Questionnaire; TSLP-human thymic stromal lymphopoietin receptor; TEC-total eosinophil count; PGD2-prostaglandin D2; FeNO-fractional exhaled nitric oxide.
Biologic anti-eosinophil drugs in asthma have recently aroused much interest. The Food and Drug Administration (FDA) has approved omalizumab, mepolizumab, reslizumab, benralizumab, dupilumab, and tezepelumab for asthma treatment. These monoclonal antibodies have been shown to improve asthma control, decrease asthma exacerbation rates, and reduce glucocorticoid dependence [231,232]. Due to the heterogeneity of the asthma clinical picture and treatment responses, identifying the appropriate patients for these drugs is crucial.
We believe that the drugs listed herein constitute a promising strategy for the treatment of severe eosinophilic asthma. However, this therapeutic approach has limitations. To date, only a few human studies and clinical trials on several reviewed drugs have been conducted in asthma. Further studies are required to corroborate their efficacy and longterm safety. Another limitation is the lack of data regarding sex, age, BMI, comorbidities, smoking status, allergic history, and disease severity in some reviewed papers. Moreover, asthma and COPD overlap syndrome accounts for 15-25% of obstructive pulmonary diseases [233][234][235][236]. We conclude that the proposed management would not be successful in those patients.

Conclusions
Asthma is a multifactorial disease with a pathogenesis involving a multitude of cytokines, chemokines, and growth factors. Approximately 65% of patients have poorly controlled asthma. Thus, there is a need to elaborate novel therapeutic and diagnostic strategies. VEGF and EDN levels are elevated in asthmatics and have a significant influence on modulating inflammatory processes. Several currently used therapeutics, e.g., budesonide and montelukast, have anti-VEGF properties. Among others with such activity are lichochalcone A, bevacizumab, diosmetin, epigallocatechin gallate, IGFBP-3, Neovastat, endostatin, PEDF, and melatonin. Azithromycin (AZT) at 500 mg for 48 weeks (3 times/week) reduces exacerbation rates and may be considered in adults with persistent symptomatic asthma. However, the long-term effects of AZT on community microbial resistance require further investigation. Vitamin D supplementation may enhance corticosteroid responsiveness. EDN is recommended to monitor asthma control status and anti-eosinophil drug therapy in children and adults. More research is needed to evaluate the efficacy of add-on therapy with listed anti-VEGF and anti-eosinophil drugs.  Data Availability Statement: Data sharing is not applicable as no datasets were generated or analysed during the current study.